Biochemical testing is becoming an increasingly important tool for various assays including, for example for detecting and monitoring the presence or absence of diseases. While tests have long been known for obtaining basic medical information such as blood type and transplant compatibility, for example, advances in understanding the biochemistry underlying many diseases have vastly expanded the number of tests which can be performed. Thus, many tests have become available for various analytical purposes, such as detecting pathogens, diagnosing and monitoring disease, detecting and monitoring changes in health, and monitoring drug therapy. Genomic data in conjunction with the ability to prepare combinatorial libraries of chemical components has facilitated the discovery of new drugs.
There has long been a need for “complete systems” allowing various stages of nucleic acid, e.g., DNA, analysis to be performed on a single device, such as a microchip. Fully integrated, high throughput systems are needed which rapidly and simultaneously perform DNA analyses such as DNA separation and PCR and thereby permit disease diagnosis or detection. Sanders, et al. (2000) Trends in Analytical Chemistry, 19(6): 364-378. Systems where up to four samples can be amplified and analyzed on the same chip have been previously disclosed. L. C. Waters, et al. (1998) Anal. Chem., 70: 5172. In addition, small, disposable mass-produced devices for conducting PCR have been reported; see e.g. U.S. Pat. No. 5,498,392. For example, Yuen, et al. (2001) Genome Research 11:405-412, provides a plexiglass-based microchip module designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module comprises a micro heater-cooler and a series of microchannels for transporting human whole blood and reagents. The white blood cells are first isolated from a small volume of whole blood in integrated cell isolation-PCR containing gate-like microstructures which retain white blood cells, albeit at a very low concentration and efficiency (i.e. 3-5%). Red blood cells pass through the micro-filters but tend to clog up the filters over time causing inefficiencies in white blood cell isolation. The Yuen, et al. microchip employs a microtemperature sensor, making the Yuen, et al. chip expensive to fabricate.
DNA microarray devices are also currently employed for DNA analysis. Two types of DNA microarray technologies are known, cDNA microarray and oligo microarray. Both technologies examine the mRNA expression in a sample based on hybridization reactions. The microarray-based assays are cumbersome, taking about a day to complete and requiring standalone equipment to conduct sequential batch analyses. Rapid diagnoses are precluded and current microarray devices do not permit sample preparation to be integrated onto the chip.
Additional disadvantages of the current on-chip DNA analysis systems have recently been reported. Such disadvantages include lack of sample injection ability, poor DNA isolation and inability to conduct multiple PCR analyses. Yuen, et al. Page 4005, right column.
Nucleic acids play a direct role in cellular processes, including those resulting in disease states by functioning in the control and regulation of gene expression. Hybridization techniques have been developed to conduct various types of nucleic acid analyses to better understand how genetic information functions in diverse types of biological processes. Hybridization methods generally employ the binding of certain target nucleic acids by nucleic acid probes under controlled conditions thereby enabling hybridization to occur only between complementary sequences. Using hybridization techniques, it is possible to conduct gene expression studies as well as a variety of other types of analysis. For example, gene expression studies are important because differential expression of genes has been shown to be associated with disease states. Many disease states have been characterized by differences in the expression of various genes either through change in copy number of the genetic DNA or through alterations in levels of transcription. In certain diseases, infection by a particular virus is characterized by elevated expression of genes.
Chips to which nucleic acid probes are attached can be used to conduct nucleic acid analyses. Probes can be attached at specific sites on the chip, such as assay stations. Assay stations are situated in areas intermediate between first and second multi-purpose channels, wherein assay reactions are run, as detailed below. In some applications, the chip may include assay stations arranged in the form of an array. Genetic methods utilizing arrays on chips are advantageous because such chips allow for simultaneous, parallel processing that can increase the rate at which analyses can be conducted as compared to conventional methods which often require labor intensive sample preparations and electrophoretic separations. Current nucleic acid methods using chips typically require complex off-chip sample DNA isolation, integrated micro-heaters and micro-temperature sensors for PCR thus making current chips and associated methods of using same very expensive and non-disposable.
It is an object of this invention to provide disposable microchips permitting multiples of assay stations for carrying out various biochemical assays in real-time.